After decades of decline in the incidence of tuberculosis, an alarming increase in new cases is occurring. Infection with M. tuberculosis, the causative agent of tuberculosis, most commonly occurs by inhalation of droplets containing only a few live bacilli. The mycobacteria replicate in lung tissue to form a primary focus of infection and from there enter the local lymphatic system. The infection then disseminates widely through the body via the blood and lymphatic system. The initial lesions usually heal to form tiny granulomas that may harbor viable tubercle bacilli indefinitely. Post-primary tuberculosis is the most common form of clinical tuberculosis and is usually pulmonary. The disease can occur many years after initial infection and is thought to be due to a temporary loss or diminishing of cell-mediated immunity (due to, for example, increasing age, illness, malnutrition, or alcoholism) leading to reactivation of dormant tubercle bacilli in lesions. Acquired Immunodeficiency Syndrome (AIDS), increased poverty and its attendant malnutrition are now important factors leading to the increased incidence of the disease.
A tentative diagnosis of tuberculosis or other mycobacteria diseases may be made on a basis of clinical grounds, radiology, and on the finding of acid-fast bacilli in smears of sputum, blood bronchial-lavage, gastric-washing, urine, or cerebral spinal fluid. Smears of these specimens typically are stained by the Ziehl-Neelsen technique or by fluorescent rhodamine-auramine dye and examined by microscopy. However, microscopically positive sputum samples are found in only about 30 to 50 percent of pulmonary tuberculosis patients and thus, cultures must always be performed. Culturing for the presence of M. tuberculosis and other mycobacterial species is a time-consuming and difficult process because some mycobacterial species are very slow-growing and have fastidious nutritional requirements. While some samples may be inoculated directly onto culture medium, most specimens require decontamination with, for example, strong alkali. Specimens are then typically inoculated onto egg-based media such as Lowenstein-Jensen medium and/or defined media such as Middlebrook 7H9 broth in the presence of antibiotics such as penicillin to inhibit growth of other bacteria. Cultures are then incubated at 35.degree. C. to 37.degree. C. for 1-7 weeks. However, even culture techniques are only about 70% effective.
Other techniques have also been developed for the detection of M. tuberculosis. These methods include nucleic acid hybridization assays using probes directed to nucleic acid sequences found in M. tuberculosis. For example, PCT Application No. WO 91/19004 entitled "Specific Detection of Mycobacterium Tuberculosis" by Guesdon and Thierry published Dec. 12, 1991 teaches the use of polynucleotide primers corresponding to those found between bases 1-30; 250-275; 1029-1058; 1200-1229; 1260-1289; 1263-1294; 1735-1764 and 1772-1796 of IS 6110, an insertion element present in multiple copies in many M. tuberculosis strains as described in Thierry et al. Nucl. Acids Res. 18:188 (1990). The application claims sequences derived from IS 6110 and sequences at least 80% homologous thereto. The probes described in that application were used for the detection of M. tuberculosis using the polymerase chain reaction.
In PCT Application No. WO 88/03957 published Jun. 25, 1988 by Hogan, et al. a method is described for the detection of non-viral organisms including M. tuberculosis and M. intracellulare using nucleic acid hybridization techniques. The method comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a region of ribosomal RNA (rRNA) selected to be unique to a particular non-viral organism or a group of non-viral organisms sought to be detected. The target rRNA is selected by comparing one or more variable region rRNA sequences of the non-viral organisms of interest with one or more variable region rRNA sequences from one or more non-viral organisms sought to be distinguished. Probe sequences which are specific for 16S rRNA variable subsequences of Mycobacterium avium, Mycobacterium intracellulare, and the Mycobacterium tuberculosis-complex bacteria and which do not cross-react with nucleic acids from each other or any other bacterial species under proper stringency are identified. A probe specific to three 23S rRNA variable region subsequences from the Mycobacterium tuberculosis-complex bacteria is also disclosed as are rRNA variable region probes useful in hybridization assays for bacteria of the genus Mycobacterium (15S, 23S rRNA specific); for Mycobacterium avium in the region corresponding to bases 185-225 of E. coli 16S rRNA; RNA of Mycobacterium intracellulare in the region corresponding to bases 185-225 of E. coli 16S RNA; to rRNA of the species included in the Mycobacterium tuberculosis complex in the region corresponding the bases 185-225 of E. coli 16S RNA; to rRNA of the species included in the Mycobacterium tuberculosis complex in the region corresponding to the bases 540-575, 1155-1190, and 2195-2235 of E. coli 23S RNA; to RNA of the genus Mycobacterium in the region corresponding to 1025-1060 of E. coli 16S RNA; and others.
U.S. Pat. No. 4,851,330 by Kohne issued on Jul. 25, 1989, addresses the use of nucleic acid hybridization to detect and quantify non-viral microorganisms. More particularly the patent describes the preparation of cDNA probes which are complementary only to ribosomal RNA subsequences known to be conserved in an organism, category or group of organisms. By way of illustration, the patent describes the production of a cDNA probe complementary to ribosomal RNA from Mycoplasma hominis but which was not complementary to human ribosomal RNA. The patent alleges that the M. hominis probes are useful in the detection of M. hominis contamination in human tissue cultures and in other cultures of mammalian cells.
U.S. Pat. No. 5,168,039 to Crawford et al. is directed to an isolated purified repetitive DNA sequence for use in detecting M. tuberculosis complex in clinical material. The patent describes the cloning and sequencing of a repetitive element found in M. tuberculosis chromosomal DNA and further describes the use of these cloned repetitive elements as probes and primers for the detection of representative strains of the M. tuberculosis complex.
U.S. Pat. No. 5,183,737 to Crawford et al. (divisional of U.S. Pat. No. 5,168,039 described above) teaches the use of the aforementioned repetitive elements as primers for the detection of bacteria of M. tuberculosis complex using the polymerase chain reaction. The patent also teaches the use of the repetitive elements as probes for the detection of bacteria of the M. tuberculosis complex using a membrane based nucleic acid hybridization assay.
PCT Application WO90/15,157 by Lane et al. published Dec. 13, 1990, addresses "universal" nucleic acid probes for eubacteria and methods for the detection of bacteria. The application describes nucleic acid probes and probe sets which hybridize under specific conditions to the ribosomal RNA molecules (rRNA), rRNA genes (rDNA), and certain amplification and in vitro transcription products thereof but which do not hybridize under the same conditions to rRNA or rDNA of eukaryotic cells which may be present in test samples. More specifically, the probes described in this application are specifically complementary to certain highly conserved bacterial 23S or 16S rRNA sequences. The probes were selected using a computer algorithm operating on aligned sets of 16S and 23S rRNA sequences to identify regions of greatest similarity among the eubacteria. Nucleic acid probes so derived hybridize most widely among diverse bacterial species. Probes found homologous among the bacteria species were also assessed for differences with non-bacterial rRNA sequences using a computer algorithm. Ultimately, 41 probes were selected based on these analysis; 22 targeting 23S rRNA and 19 targeting 16S rRNA. The 16S amplification primers described in that application include primers which detect most eubacteria, Borrelia and spirochetes, the enterics, Deinococcus, Campylobacter, and the Fusobacteria and Bacillus species. Several of these probes are assertedly capable of detecting Mycobacteria kansasii and M. bovis. The application describes the use of sandwich type hybridization assays for the detection of hybridization between probes and rRNAs in samples suspected of containing bacteria and also describes the use of the polymerase chain reaction (PCR) directed to 16S rRNAs using probes as derived above. The probes described in that application (both 16S, and 23S rRNA probes) were assertedly able to detect a wide variety of bacterial species.
PCT application WO90/12,875 by Hance et al. published Nov. 1, 1990 discloses nucleotide sequences of actinomycetales and their application to the synthesis or detection of nucleic acids found in actinomycetales. The application discloses a 383 base pair polynucleotide coding for the 65 kD (kilodalton) mycobacterial antigen which has homologs in 8 species of mycobacteria including M. tuberculosis, M. avium, M. fortuitum, M. paratuberculosis, BCG, M. kansasii, M. malmoense, and M. marinum. The use of these probes for the detection of DNA and/or the products of transcription of these bacteria is described. More particularly, an oligonucleotide comprising bases 397 through 416 of the 65 kD antigen is disclosed (See, Shinnick et al. Infect. and Immun. 56:446-451 (1988)). Another sequence corresponding to bases 535 through 554 of the same gene coding for the 65 kD antigen is also disclosed. The application proposes the use of the aforementioned oligonucleotides as primers in the polymerase chain reaction directed toward the detection of mycobacterial species.
European Patent Application No. 0 395 292 by Barry et al. published on Oct. 31, 1990, describes a method for generating DNA probes specific for various microorganisms. Specific probes are disclosed for Aeromonas hydrophila, Aeromonas salmonicida, Clostridium difficile, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium avium, Salmonella typhimurium, and other bacteria. A DNA probe for M. avium was obtained from a variable intergenic region intermediate the genes coding for 16S ribosomal RNA and 23S ribosomal RNA. DNA probes for M. bovis were obtained from a variable intergenic region intermediate the 16S ribosomal RNA gene and the 23S ribosomal rRNA gene and from the V6 variable region of the gene coding for 16S ribosomal RNA. Similarly, a DNA probe for M. tuberculosis was obtained from the variable intergenic region intermediate the 16S and 23S ribosomal RNA genes.
The nucleotide sequence of Protein Antigen B gene (pab gene) of Mycobacterium tuberculosis was described by Anderson and Hanson, Inf. and Immun. 57:2481-2488 (1989). The pab gene is 1993 nucleotides in length. The deduced amino acid sequence of the pab gene reveals 30% homology to a phosphate-binding protein (Ps+S) from E. coli. Protein antigen b was selected for analysis because of its association with virulent strains of M. tuberculosis and, to a lesser extent, with M. bovis BCG and because BCG has been used with some success as a vaccine against tuberculosis. See, e.g., Hart, P. D. and Sutherland, I., British Medical Tuberculosis Journal 2:293-295 (1977).
Baird et al. J. Gen. Microbiol. 135:931-939 (1989) have cloned and sequenced the 10 kD antigen gene of Mycobacterium tuberculosis. The 10 kD antigen has been implicated (by analogy to the 10 kD protein of M. bovis) in the induction of T-cell mediated delayed type hypersensitivity. The 10 kD antigen gene was isolated using a DNA probe corresponding to the N-terminal amino acid sequence of the M. bovis 10 kD antigen. This probe was selected because of the demonstrated immunological cross-reactivity between the 10 kD M. bovis antigen and the 10 kD antigen of M. tuberculosis. Cloning and sequencing of the M. tuberculosis 10 kD antigen gene revealed a coding sequence of 300 nucleotides which encode 99 amino acids having an aggregate molecular weight of 10.7 kD. The sequence was shown to be homologous to two procaryotic heat-shock proteins and additionally to have heat-shock like promoter sequences upstream from the initiation codon.
Rogall et al. Int. J. Syst. Bact. 40:323-330 sequenced and analyzed the 16S rRNA genes of M. tuberculosis, M. bovis, M. bovis BCG, M. tuberculosis H37, M. marinum, M. kansasii DSM 43224, M. simiae ATCC 25275, M. scrofulaceum ATCC 19981, M. szulgai ATCC 25799, M. gordonae ATCC 14470, M. xenopi ATCC 19250, M. flavescens ATCC 14474, M. avium DSM 43216, M. intracellulare ATCC 15985, M. paratuberculosis ATCC 19698, M. gastrae ATCC 15754, M. malmoense ATCC 29571, M. nonchromogenicum ATCC 19530, M. terrae ATCC 15755, M. chelonae ATCC 14472, M. smegmatis ATCC 14468, M. fortuitum ATCC 6841 and Nocardia asteroides ATCC 3306. The data obtained from this analysis revealed the phylogenetic relationships between the bacteria. The data showed that the fast growing Mycobacterium, M. fortuitum, M. chelonae, M. smegmatis and M. flavescens formed a distinct group separate from all of the other mycobacteria tested. All of the slow-growing mycobacteria species were highly related having similarity values greater than 94.8%.
Hermans et al. Inf. and Imm. 59:2695-2705 (1991) determined the sequence of the single copy insertion element IS987 from M. bovis BCG. The sequence of IS987 was noted to be virtually identical to the insertion sequence IS986 from M. tuberculosis and to reside in a region of the M. bovis BCG chromosome containing 20 identical copies of a 36 bp direct repeat, each separated by 35-41 bp of spacer DNA. The data also indicated that IS987 is nearly identical to IS6110 from M. tuberculosis, differing in that IS987 has an open reading frame (ORF) designated ORFa in a single ORF whereas IS6110 contains ORFa composed of two different ORFs. These open-reading frames may play a role in the expression of a putative transposase. The data also show that the size and composition of the direct-repeat containing region of the M. tuberculosis chromosome is polymorphic and that these repeats were not found in nine other mycobacterial species tested. The authors also suggested that the polymorphism seen in the IS insertion sites allow the easy typing of strains of M. tuberculosis complex by restriction fragment length polymorphism analysis.
Thierry et al., Nucl. Acids Res. 18:188 (1990) cloned and sequenced the insertion sequence IS6110 from M. tuberculosis. The DNA sequence of 1361 nucleotides showed characteristics of insertion sequence (IS) including inverted repeat sequences which are (28 bp in length with 3 mismatched bps and direct 3 bp repeats at each end of the IS element. The authors suggest that this IS element will be useful as a probe for the identification of the M. tuberculosis complex.
As noted above, oligonucleotide probes are used for the detection of various microorganisms by means of nucleic acid hybridization using techniques such as dot blot, slot blot, Southern blots, solution hybridization, in situ hybridization and others. Alternatively, the polymerase chain reaction may be used which amplifies the target DNA. U.S. Pat. No. 4,683,195 to Mullis et al. describes the details of the polymerase chain reaction.
Of interest to the background of the invention, is an alternate mechanism for target amplification known as ligase chain reaction (LCR). In LCR, probe pairs are used which include two primary probe partners (first and second probes) and two secondary probe partners (third and fourth) all of which are employed in excess. The first probe of a probe pair hybridizes to a first segment of the target strand and the second probe of a probe pair hybridizes to a second segment of the same target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3' hydroxyl relationship and so that a ligase can covalently fuse or ligate the two probes of the pair into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a potion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes will also hybridize to the target complement in the first instance. Once the fused strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary fused product. It is important to realize that the fused products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in K. Backman, et al. EP-A-320 308 published Jun. 14, 1989 and K. Backman, et al., EP-A-439 182 published Jul. 31, 1991, both incorporated by reference in their entirety.
A potential problem associated with ligase chain reaction is background signal caused by target independent ligation of the probes. Since the third probe hybridizes to the first probe and the fourth probe hybridizes to the second probe, the probes, which are added in excess, can easily form duplexes among themselves. These duplexes can become ligated independently of the presence of target to form a fused product which is then indistinguishable from the desired amplified target, yet which is still capable of supporting further amplification. Although target independent blunt-end ligation of these duplexes is a relatively rare event, it is sufficiently common to cause undesirable high background signals in diagnostic assays.
Some attempts to overcome this background problem have been published. For example, WO 90/01069 (Segev Diagnostics) and GB 2 225 112 A (Imperial Chemical Industries Plc.) describe versions of a ligation-based amplification scheme which includes a polymerase-mediated gap-filling step prior to ligation. In addition, EP-A-439 182 to K. Backman et al. published Jul. 31, 1991, teaches variations of LCR that reduce background. One such variation involves gap filling and ligation.
In the gap-filling ligation method described in EP-A-439 182 to Backman et al. published Jul. 31, 1991, instead of using probe pairs capable of forming blunt-ended duplexes, at least one probe of a probe pair initially includes a "modified" end which renders the resultant duplex "non-blunt" and/or not a substrate for the ligase catalyzed fusion of the probe pair. A "modified" end is defined with respect to the point of ligation rather than with respect to its complementary probe. A "modified end" has omitted bases to create a "gap" between the terminus of one probe of a probe pair and the terminus of the other probe of a probe pair. Other modifications include mismatches between a probe and a target sequence. "Correction" of the modification is then carried out to render the probes ligatable. "Correction" refers to the process of rendering (in a target dependent manner), the two probes of a probe pair ligatable. Thus, only those probes hybridized to target, target complement or polynucleotide sequences derived therefrom, are corrected. "Correction" can be accomplished in a variety of ways depending on the type of modified end used.
There continues to exist a need in the art for new reagents and methods for the rapid and sensitive detection of M. tuberculosis and for bacteria of the genes Mycobacterium.